Resistance to bacteriophage incurs a cost to virulence in drug-resistant Acinetobacter baumannii

Abstract Introduction. Acinetobacter baumannii is a critical priority pathogen for novel antimicrobials (World Health Organization) because of the rise in nosocomial infections and its ability to evolve resistance to last resort antibiotics. A. baumannii is thus a priority target for phage therapeutics. Two strains of a novel, virulent bacteriophage (LemonAid and Tonic) able to infect carbapenem-resistant A. baumannii (strain NCTC 13420), were isolated from environmental water samples collected through a citizen science programme. Gap statement. Phage-host coevolution can lead to emergence of host resistance, with a concomitant reduction in the virulence of host bacteria; a potential benefit to phage therapy applications. Methodology.In vitro and in vivo assays, genomics and microscopy techniques were used to characterize the phages; determine mechanisms and impact of phage resistance on host virulence, and the efficacy of the phages against A. baumannii. Results.A. baumannii developed resistance to both viruses, LemonAid and Tonic. Resistance came at a cost to virulence, with the resistant variants causing significantly reduced mortality in a Galleria mellonella larval in vivo model. A replicated 8 bp insertion increased in frequency (~40 % higher frequency than in the wild-type) within phage-resistant A. baumannii mutants, putatively resulting in early truncation of a protein of unknown function. Evidence from comparative genomics and an adsorption assay suggests this protein acts as a novel phage receptor site in A. baumannii. We find no evidence linking resistance to changes in capsule structure, a known virulence factor. LemonAid efficiently suppressed growth of A. baumanni in vitro across a wide range of titres. However, in vivo, while survival of A. baumannii infected larvae significantly increased with both remedial and prophylactic treatment with LemonAid (107 p.f.u. ml–1), the effect was weak and not sufficient to save larvae from morbidity and mortality. Conclusion. While LemonAid and Tonic did not prove effective as a treatment in a Galleria larvae model, there is potential to harness their ability to attenuate virulence in drug-resistant A. baumannii.

Figure S4.Pairwise comparison of LemonAid and Tonic: 87% nucleotide pairwise identity across length, highlighting differences across genes (Geneious, Mauve, (Darling et al., 2004)) Figure S5.Viridic intergenomic similarity of LemonAid and Tonic against the 13 most similar phage genomes when searched against the LemonAid genome using NCBI Blast.The number indicates % intergenomic similarity, anything <95% is considered a separate species.Thus, LemonAid and Tonic are two variants of the same novel species.
Figure S6.Distribution of two prophage, Fizzy and Cloudy, that were induced from host A. baumannii NCTC 13420 when infected with the virulent phage LemonAid.The tree is mid-point rooted.Fizzy is marked in red, Cloudy in green.It is worth noting that the phages appear to share the same attB site, integrating just downstream of the 6S RNA (ssrS) gene.Interestingly, Cloudy creates a 60 bp repeat at both ends of the prophage region that overlaps the ssrS sequence.It is possible this enables the second clade of prophages (e.g Fizzy) to integrate at the right repeat sequence, hence we observe tandem insertion of two prophages.There was no difference between phage treatment (green) and A. baumannii positive control (magenta).Note: data collection was not taken between 12 and 24 hours.Kaplan Meier survival curve and model χ 2 = 89.6,3d.f, p = 0.001, corrected p-value for pairwise comparison between A. baumannii positive control and LemonAid treated larvae = 0.23, ns).

Supplementary tables
Table S1.Treatment groups and injection protocol for efficacy of LemonAid against A. baumannii.

Method S1 -Plaque assays and producing an axenic phage
Plaque assays: The soft-agar overlay technique (Kropinski et al., 2009) was used to determine if any A. baumannii phage were present in the enriched samples.Briefly, 30mL of 1% LB bottom agar, containing CaCl2 and MgCl2 at a final concentration of 10mM, was poured into a petri-dish, 3mL of 0.65% LB top agar was mixed with 1mL exponential phase (0.6 OD600) A. baumannii (strain NCTC 13420) and 1mL of sample filtrate, and poured onto the solidified bottom agar plate and allowed to cool.Plates were incubated overnight at 37°C and then examined for the presence of plaques.Cores of the resulting plaques were taken and suspended in 100µL SM buffer (containing 100 mM NaCl, 50 mM Tris-HCl; 8 mM MgSO4•H2O).Core suspensions were serially diluted 1:10 and 10µl of each dilution was spotted onto 0.65% top agar LB seeded with 1mL 0.6 OD600 A. baumannii, using the overlay method.A core of a solitary plaque was taken, suspended in SM buffer and serially diluted.
This process was repeated three times before selecting a plaque to take forward in experiments, to ensure the phages were axenic.
-Spot assays to determine resistance to phage Bottom agar plates were poured and set as described above.1mL of A. baumannii culture at 0.6 OD600 was added to 3mL of warm, liquid 0.65% LB top agar and poured onto the solidified bottom agar plate.After the top agar had been allowed to set, 10 µL of phage lysate were spotted onto the top agar and allowed to dry, before being incubated overnight at 37°C, and plaques counted the next day.To assess the distribution of prophage clades represented by Fizzy and Cloudy, BLASTN was performed against a dataset of 420 complete Acinetobacter baumannii genomes where putative prophage regions had been predicted using PhiSpy (https://github.com/linsalrob/PhiSpy,Turner, unpublished data).Presence of either phage was determined using a strict inclusion threshold of ≥95 sequence similarity.These data were used to annotate a core genome phylogeny created using FastTree with 100 bootstraps from a MAFFT (Katoh & Standley, 2013)core gene alignment produced by Roary (Page et al., 2015).Using a recombination naïve approach, Pasteur sequence type was predicted for each genome using mlst (https://github.com/tseemann/mlst)and the tree was annotated using ITOL (Letunic & Bork, 2021) .

Method S3 -Short and long read sequencing
We extracted bacterial DNA from 1mL of wildtype and phage-resistant variants of A.
baumannii using the Circulomics© Nanobind high molecular weight genomic DNA extraction kit.We used Oxford Nanopore's Rapid Sequencing kit (RAD004) to produce libraries as per the manufacturer's instructions.We primed a SpotON flow cell (FLO-MIN106D R9) using the flow cell priming kit (EXP-FLP002), and sequenced the library on a MinION Mk1B.Data acquisition was achieved in real time using MinKnow v.# and basecalling was carried out using guppy basecaller (Oxford Nanopore).
In parallel, short read sequencing was carried out by the Exeter sequencing Service.Libraries were prepped using Nebnext library prep and sequenced on the NovaSeq (Illumina).

Method S4. Search for evidence of function of hypothetical protein at 2,467,798 bp in the A. baumannii genome
We used the following tools:

Method S5 -Preparation of phage and bacterial inoculums for galleria assays Bacterial strains and preparation of bacterial inoculum
A. baumannii reference strain NCTC 13420 cells were stored as cryostocks at -80°C, and cultured in LB broth or agar aerobically at 37°C.From streak plates, 20 individual colonies were selected and inoculated into 15mL LB containing CaCl2 and MgCl2 at a final concentration of 10mM, and incubated at 37°C on an orbital shaker at 200rpm for ~2 hours, until OD600 = 0.6 (~2 hours).Cultures were centrifuged at 4000rpm for 10 minutes, disposed of the supernatant and re-suspended the pellet in 5ml of 1xPBS.The bacterial suspension was checked and diluted if necessary to OD590 = 1 ±0.05.Bacterial colony counts of the inoculum were confirmed on LB agar: mean CFU/mL = 4.8 x 10 8 (ranging from 4.0 to 5.7 x 10 8 CFU/mL across assays) and used this dose in the infection assays.

Preparation and quantification of phage inoculum
Before its use as an inoculum, 1mL phage lysate was re-filtered (0.22nm filter) to ensure no host cells were present.As an additional control, phage solution was spotted out onto a LB agar plate and grown at 37 degrees overnight, to ensure no bacterial cells had contaminated it.We carried out PFU counts and determined the concentration of LemonAid and Tonic to be 5.3 x10 8 and 6.63 X 10 9 PFU/mL respectively.Preliminary experiments revealed that 5.3 x 10 8 PFU/mL phage solution caused melanisation in 50% (N=10) of larvae within 24 hours, compared to no melanisation in control larvae that were inoculated with LB buffer (N=10) or PBS (N=10).No death or malaise resulted from inoculation with 10 7 PFU/mL, thus we used this concentration in all subsequent experiments.

Method S6 -Galleria larvae inoculations and controls
Research-grade G. mellonella larvae in their final-instar stage were purchased from Biosystems Technology, TruLarv™.Larvae were stored in the dark at 15°C and used within the week of arrival.The needle was prepared before and between treatments by washing twice in 70% ethanol and twice in 1x PBS; needles were checked for sterility before and after injections by spotting PBS on LB agar, incubating at 37°C overnight and checking for colonies after 24 hours.Separate needles were used for bacteria and phage inoculums.

Method S7 -Endotoxin testing
The endotoxin concentration of the phage preparation was determined using the ToxinSensor chromogenic lyophilised amoebocyte lysate (LAL) endotoxin assay kit (GenScript, New Jersey, USA).The assay was carried out according to the manufacturer's instructions, using one-tenth volumes, in a pyrogen free round bottomed 96 well plate (Grenier Bio-One, Kremsmünster, Austria).Endotoxin standards over the range 0.0 -1.0 EU/mL were prepared in the LAL reagent water provided.The phage preparation was diluted to 10 -8 in cell culture grade sodium chloride solution (0.9%, Sigma-Aldrich, St. Louis,

Figure
Figure S3.a) Annotated LemonAid and b) Tonic genome using Pharokka

Figure S7 .
Figure S7.Protein of unknown function in full (green) and truncated (blue) when an 8 bp insertion is present

Table S3 .
Results of informatics search on hypothetical protein of interest Structure found but the protein on which it is based has no known function

Table S4 .
Full linear model showing the effect of treatment, time and batch on melanisation of larvae in the phage-resistant assay, where the reference group is A. baumannii positive control

Table S5 .
Full linear model showing the effect of treatment, time, batch and experiment on melanisation of larvae in the phage treatment assay, with PBS negative control as reference group.

Table S7 .
Full model of melanisation for dose-response assay, with the negative PBS control as the reference group.